Regulation Mechanism Of Rapamycin On The Growth And Infection Of Botryosphaeria Dothidea

Rapamycin is a macrolides antibiotic produced by Streptomyces hygroscopicus,which had been documented as its high antifungal activity.Rapamycin regulates multiple cell metabolism pathways by inhibiting the protein called target of rapamycin（TOR）.It is of great interest to further explore the mechanism.Botryosphaeria dothidea,a fungal pathogen causing the devastive trunk canker on Carya cathayensis,was selected in this study.The inhibitory effect of rapamycin on the growth of B.dothidea were determined.The regulatory of rapamycin on the nitrogen response and infection of B.dothidea were investigated.The mechanism of rapamycin on this fungus was analyzed.The main results were listed as following.1.The inhibition effect of rapamycin on B.dothidea was measured by hyphal growth rate method.The results showed that the hyphal growth was significantly reduced after rapamycin treatment for 5days.The 50%effective concentration(EC₅₀)value was 5.00×10^-3μg/m L,which was significantly lower than that of azoxystrobinon（6.32μg/m L）.Meanwhile,inhibitory effect of rapamycin on hyphal biomass accumulation of B.dothidea was measured.It was found that the accumulation of hyphal biomass was significantly reduced after rapamycin treatment for 4 days,and the EC₅₀value was2.00×10^-3μg/m L.The results indicated that rapamycin exhibited strong inhibitory effect on the hyphal growth of B.dothidea.2.The hyphal cell of B.dothidea treated with rapamycin were observed under light microscopy and scanning electron microscopy（SEM）.The hypha were cultured amended with 5.00×10^-3μg/m L rapamycin for 4 day.Microscopic examination revealed that B.dothidea hyphae treated with rapamycin were more highly branched and septate when compared to the untreated hyphae.In addition,when viewed under SEM,the hyphae treated with rapamycin aged prematurely and were wrinkled,while the hyphae treated with the control were normal.It is speculated that rapamycin induces the apoptosis of hyphal cells.3.The effect of rapamycin on the lipid droplet formation in hyphal cell of B.dothidea was determined by oil red O staining method.The results showed that addition of rapamycin significantly promoted the accumulation of lipid droplets.Comparing to the control,the average diameter of lipid droplets treated with 5.00×10^-3μg/m L rapamycin for 5 day increased by 11 times,and the number of lipid droplets per cell decreased by 66.6%.Higher concentration of rapamycin resulted in more accumulation of lipid droplet.The results indicated that rapamycin might induce the lipid starvation response of the pathogen.4.The cellophane penetration method was used to evaluate the regulation of rapamycin on the nitrogen response and hyphal infection of B.dothidea.It was observed that various nitrogen sources affected the penetration of B.dothdiea via rapamycin in the different levels.It is of interest that ammonium inhibited the hyphal invasion,while nitrate nitrogen had no inhibitory effect.Rapamycin reduced significantly the inhibition of ammonium on the penetration of B.dothidea.The results indicated that rapamycin might function in regulation of nitrogen response and fungal infection of B.dothidea.5.A high-quality draft genome sequence of a strain CK 16 derived from a single conidium culture of B.dothidea was provided.The nuclear genome consists of 1 303 scaffolds with a total assembly length of 44.39 Mbp(N₅₀=354 836 bp),54.60%GC-content,and a maximum scaffold size of 1 483403 bp.In total,538 glycoside hydrolase genes and 46 genes related to toxin synthesis,such as NRPS、PKS、DMATS were located.6.Transcriptome analysis was used to determine the differences of rapamycin on the gene expression of B.dothidea.The hypha of B.dothidea were treated by 5.00×10^-3μg/m L rapamycin for1day,and then the m RNA were extracted and assayed.In total,1 308 genes were changed in transcriptional level,which includes 532 up-regulated genes and 776 down-regulated genes.It was further found that rapamycin negatively regulated the putative pathogenesis-determined genes（Bd Sch9,Bd Ppg1,Bd Sitf1,Bd Imp1）and the autophagy related genes（Bd ATG1,Bd ATG5,Bd ATG7,Bd ATG17）.It was speculated that rapamycin might regulate the cell autophagy,lipid droplet formation,and pathogenicity via TOR.In summary,the inhibitory regulation of rapamycin on B.dothidea was determined in this study.Rapamycin could induce the fungal cell apoptosis and lipid droplet formation.Rapamycin also regulate the nitrogen response and fungal infection via TOR.The results in this study would contribute to mechanism research of rapamycin on cell biology and provide a novel insight of Botrysphaeria canker disease on woody plants.